Mike Westby

Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

ABSTRACT Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

Reduced Maximal Inhibition in Phenotypic Susceptibility Assays Indicates that Viral Strains Resistant to the CCR5 Antagonist Maraviroc Utilize Inhibitor-Bound Receptor for Entry

ABSTRACT Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Δ32/Δ32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

Subgroup analyses of maraviroc in previously treated R5 HIV-1 infection.

BACKGROUND We conducted subanalyses of the combined results of the Maraviroc versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients (MOTIVATE) 1 and MOTIVATE 2 studies to better characterize the efficacy and safety of maraviroc in key subgroups of patients. METHODS We analyzed pooled data from week 48 from the two studies according to sex, race or ethnic group, clade, CC chemokine receptor 5 (CCR5) delta32 genotype, viral load at the time of screening, the use or nonuse of enfuvirtide in optimized background therapy (OBT), the baseline CD4 cell count, the number of active antiretroviral drugs coadministered, the first use of selected background agents, and tropism at baseline. Changes in viral tropism and the CD4 count at treatment failure were evaluated. Data on aminotransferase levels in patients coinfected with hepatitis B virus (HBV) or hepatitis C virus (HCV) were also analyzed. RESULTS A treatment benefit of maraviroc plus OBT over placebo plus OBT was shown in all subgroups, including patients with a low CD4 cell count at baseline, those with a high viral load at screening, and those who had not received active agents in OBT. Analyses of the virologic response according to the first use of selected background drugs showed the additional benefit of adding a potent new drug to maraviroc at the initiation of maraviroc therapy. More patients in whom maraviroc failed had a virus binding to the CXC chemokine receptor 4 (CXCR4) at failure, but there was no evidence of a decrease in the CD4 cell count at failure in such patients as compared with those in whom placebo failed. Subanalyses involving patients coinfected with HBV or HCV revealed no evidence of excess hepatotoxic effects as compared with baseline. CONCLUSIONS Subanalyses of pooled data from week 48 indicate that maraviroc provides a valuable treatment option for a wide spectrum of patients with R5 HIV-1 infection who have been treated previously. (ClinicalTrials.gov numbers, NCT00098306 and NCT00098722.)

Emergence of CXCR4-Using Human Immunodeficiency Virus Type 1 (HIV-1) Variants in a Minority of HIV-1-Infected Patients following Treatment with the CCR5 Antagonist Maraviroc Is from a Pretreatment CXCR4-Using Virus Reservoir

ABSTRACT Antagonists of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-1-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for 10 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 51 of whom experienced an HIV RNA reduction from baseline of >1 log10 copies/ml, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day 11. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope (Env) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.

Maraviroc versus Efavirenz, Both in Combination with Zidovudine-Lamivudine, for the Treatment of Antiretroviral-Naive Subjects with CCR5-tropic HIV-1 Infection

BACKGROUND The MERIT (Maraviroc versus Efavirenz in Treatment-Naive Patients) study compared maraviroc and efavirenz, both with zidovudine-lamivudine, in antiretroviral-naive patients with R5 human immunodeficiency virus type 1 (HIV-1) infection. METHODS Patients screened for R5 HIV-1 were randomized to receive efavirenz (600 mg once daily) or maraviroc (300 mg once or twice daily) with zidovudine-lamivudine. Coprimary end points were proportions of patients with a viral load <400 and <50 copies/mL at week 48; the noninferiority of maraviroc was assessed. RESULTS The once-daily maraviroc arm was discontinued for not meeting prespecified noninferiority criteria. In the primary 48-week analysis (n = 721), maraviroc was noninferior for <400 copies/mL (70.6% for maraviroc vs 73.1% for efavirenz) but not for <50 copies/mL (65.3% vs 69.3%) at a threshold of -10%. More maraviroc patients discontinued for lack of efficacy (11.9% vs 4.2%), but fewer discontinued for adverse events (4.2% vs 13.6%). In a post hoc reanalysis excluding 107 patients (15%) with non-R5 screening virus by the current, more sensitive tropism assay, the lower bound of the 1-sided 97.5% confidence interval for the difference between treatment groups was above -10% for each end point. CONCLUSIONS Twice-daily maraviroc was not noninferior to efavirenz at <50 copies/mL in the primary analysis. However, 15% of patients would have been ineligible for inclusion by a more sensitive screening assay. Their retrospective exclusion resulted in similar response rates in both arms Trial registration. ClinicalTrials.gov identifier: (NCT00098293) .

A Double-Blind, Placebo-Controlled Trial of Maraviroc in Treatment-Experienced Patients Infected with Non-R5 HIV-1

BACKGROUND Maraviroc, a CCR5 antagonist, is active against R5 but not X4 or dual- or mixed-tropic strains of human immunodeficiency virus type 1 (HIV-1). A phase 2b study was conducted to determine the safety and efficacy of maraviroc in combination with optimized background therapy in treatment-experienced patients infected with dual- or mixed-tropic HIV-1. METHODS Treatment-experienced patients with an HIV-1 RNA level 5000 copies/mL who had received 3 classes of drugs and/or were infected with virus resistant to 2 drug classes and were infected with non-R5 HIV-1 were randomized to receive optimized background therapy plus maraviroc (once or twice daily) or placebo. The primary end point was change in HIV-1 RNA level from baseline to 24 weeks. RESULTS Among 167 patients infected with dual- or mixed-tropic HIV-1, baseline mean HIV-1 RNA levels were >5 log(10) copies/mL and median CD4(+) cell counts were <50 cells/microL. From baseline to 24 weeks, patients who received placebo demonstrated a mean decrease in HIV-1 RNA levels of 0.97 log(10) copies/mL, compared with mean decreases of 0.91 and 1.20 log(10) copies/mL for those who received maraviroc once (P =.83) or twice (P +.38) daily, respectively. Mean increases in CD4(+) cell counts from baseline were 36 cells/microL for patients who received placebo, 60 cells/microL among patients who received maraviroc once daily, and 62 cells/microL among patients who received maraviroc twice daily. The incidences of serious adverse events were similar among groups. CONCLUSIONS In this exploratory study involving extensively treatment-experienced patients with advanced, non-R5 HIV-1 infection, neither superiority nor noninferiority was statistically demonstrated for either maraviroc dosage compared with placebo at 24 weeks of treatment. TRIAL REGISTRATION Clinicaltrials.gov identifier NCT00098748 .

CCR5 Antagonists: Host-Targeted Antivirals for the Treatment of HIV Infection

The human chemokine receptors, CCR5 and CXCR4, are potential host targets for exogenous, small-molecule antagonists for the inhibition of HIV-1 infection. HIV-1 strains can be categorised by co-receptor tropism — their ability to utilise CCR5 (CCR5-tropic), CXCR4 (CXCR4-tropic) or both (dual-tropic) as a co-receptor for entry into susceptible cells. CCR5 may be the more suitable co-receptor target for small-molecule antagonists because a natural deletion in the CCR5 gene preventing its expression on the cell surface is not associated with any obvious phenotype, but can confer resistance to infection by CCR5-tropic strains — the most frequently sexually-transmitted strains. The current leading CCR5 antagonists in clinical development include maraviroc (UK-427,857, Pfizer), aplaviroc (873140, GlaxoSmithKline) and vicriviroc (SCH-D, Schering-Plough), which have demonstrated efficacy and tolerability in HIV-infected patients. Pharmacodynamic data also suggest that these compounds have a long plasma half-life and/or prolonged CCR5 occupancy, which may explain the delay in viral rebound observed following compound withdrawal in short-term monotherapy studies. A switch from CCR5 to CXCR4 tropism occurs spontaneously in approximately 50% of HIV-infected patients and has been associated with, but is not required for, disease progression. The possibility of a co-receptor tropism switch occurring under selection pressure by CCR5 antagonists is discussed. The completion of ongoing Phase IIb/III studies of maraviroc, aplaviroc and vicriviroc will provide further insight into co-receptor tropism, HIV pathogenesis and the suitability of CCR5 antagonists as a potent new class of antivirals for the treatment of HIV infection.

Small Molecules Targeting Hepatitis C Virus-Encoded NS5A Cause Subcellular Redistribution of Their Target: Insights into Compound Modes of Action

ABSTRACT The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

Small-Molecule Inhibitors of the LEDGF/p75 Binding Site of Integrase Block HIV Replication and Modulate Integrase Multimerization

ABSTRACT Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.

HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less efficient mechanism of gp120-CCR5 engagement that attenuates macrophage-tropism

ABSTRACT Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop–CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.